Screening Methods of Diuretic activity

Screening Methods of Diuretic activity

Classification of Screening Methods of Diuretic activity

 In vivo methods      

• Diuretic activity in rats  (LIPSCHITZ TEST)
• Saluretic activity in rats 
• Clearance method
• Stop flow technique.
• Micropuncture technique

In vitro method

• Isolated tubule Preparation
• Carbonic anhydrase inhibition
• Patch clamp technique

In vivo Methods

1)   DIURETIC ACTIVITY IN RATS (LIPSCHITZ)

PURPOSE: Test is based on water and sodium excretion in test animals and compared to rats treated with a high dose of urea.

v “Lipschitz-value” is the quotient between excretion by test animals and excretion by the urea control.

Metabolic cage for screening test 

PROCEDURE:

Ø Male Wistar rats weighing 100–200 g are used and 3 animals per group are placed in metabolic cages

q Metabolic cages:

Ø Wire mesh at bottom

Ø Funnel to collect urine.

Ø Stainless steel sieves are placed into funnel.

Ø The rats are fed with standard diet and water.

Ø 15 hr. before experiment, food and water are withdrawn.

For screening procedures 

1. Test =2 group(6 animals)

The test compound is given orally at a dose of 50 mg/kg in 5.0 ml water/kg body weight

2. Control = 2 group (6animals)

orally 1 g/kg urea  5 ml of 0.9% NaCl solution per 100 g body weight are given by gavage

Metabolic Cage for screening tests.

Ø Urine excretion is recorded upto 5hr and  24 h. 

Ø The Na+ content of the urine is determined by flame photometry & urine vol. excreted calculated for each group. 

Ø Active compounds are tested again with lower doses.

EVALUATION:

v Results are expressed as the “Lipschitz-value”, i.e., the ratio T/U, in which T is the response of the test compound, and U, that of urea treatment.

  LIPSCHITZ value :   Urine output in test animal 

                      Urine output in std drug treated animal.

            Lipschitz value  > 1.0 = positive effect.

            Lipschitz value  > 2.0 = Potent diuretic activity.

v For studying prolonged effect, 24 hr urine sample collected and analyzed.

v Dose-response curves can be established using various doses.

v Saluretic drugs,

     hydrochlorothiazide  =  1.8,

     loop diuretics    >   4.0

2)   SALURETIC ACTIVITY IN RATS      

                        

PURPOSE:

v  Excretion of electrolytes is as important as the excretion of water for the treatment of peripheral edema, CHF, hypertension. Potassium loss has to be avoided.

v So need to develop, diuretic with saluretic and K+ sparing effect.

v Diuresis test in rats is designed to determine Na+, K+, Cl-, water content and osmolarity of urine.

v Ratios between electrolytes can be calculated indicating carbonic anhydrase inhibition or a K+ sparing effect.

PROCEDURE:

Ø Male Wistar rats weighing 100–200 g fed with standard diet and water.

Ø  15 hours prior to the test, food is withdrawn but not water.

Ø 3 animals are placed in one metabolic cage and 2 groups of 3 animals

Ø Test compounds are applied in a dose of 50 mg/kg orally in 0.5 ml/100 g body weight starch suspension.

Ø Urine excretion is registered every hour up to 5 h and collected urine is analyzed by flame photometry for Na+ and K+ and Cl-

Ø To evaluate compounds with prolonged effects the 24 h urine is collected and analyzed.

Ø Furosemide, hydrochlorothiazide, triamterene, or amiloride are used as standards.

EVALUATION:

v For saluretic activity:

                            Na+ + Cl- excretion calculated.

v For natriuretic activity : Na+   is calculated.

                                                  K+

             Natriuretic effect >2

             K+ sparing effect >10.

v For estimating Carbonic anhydrous inhibition.

         

   is calculated

   

                          

• Carbonic anhydrase inhibition can be excluded at ratios between 1.0 and 0.8. With decreasing ratios slight to strong carbonic anhydrase inhibition can be assumed.

3)   STOP FLOW TECHNIQUE

PRINCIPLE:

Ø Useful in the localization of transport processes along the length of the nephron.

Ø During clamping of the ureter, glomerular filtration rate is grossly reduced.

Ø The contact time for the tubular fluid in the respective nephron segments increases, and the concentration of the constituents of tubular fluid should approximate the static-head situation.

Ø After release of the clamp, the rapid passage of the tubular fluid should modify the composition of the fluid only slightly.

Ø Urine is sampled sequentially …..

PROCEDURE :

Ø This method can be performed in different animals during anesthesia .

Ø The ureter of an animal is clamped for several minutes allowing a relatively static column of urine to remain in contact with the various tubular segments for longer than the usual periods of time.

Ø Then the clamp is released, and the urine is sampled sequentially.

Ø Small serial samples are collected rapidly, the earliest sample representing fluid which had been in contact with the most distal nephron segment.

Ø Substances examined are administered along with inulin before the application of urethral occlusion.

EVALUATION :

v In each sample the concentration of inulin, and the concentration of the substance under study are measured.

v  Fractional excretion of the substance and the glomerular marker(Inulin) are plotted versus the cumulative urinary volume. 

4)   CLEARANCE METHOD

PRINCIPLE:

Ø Indirect methods for the evaluation of renal function and provide information on the site of action of diuretics and other pharmacological agents within the nephron.

Drugs acting on	CH2O & TCH20
PCT	Increases both CH2O & TCH20
LOH	Impairs both CH2O & TCH20
DCT	Reduces CH2O but not  TCH20

Where,

C_H2O : clearance of solute free water during diuresis,

T_CH20 : reabsorption of solute free water during restriction.

PROCEDURE:

Ø Test may be performed in species from which urine and plasma can be readily collected.

Ø Experiments are performed in anaesthetized beagle dogs under conditions of water diuresis and hydropenia.

1.   Water diuresis.

    oral administration of 50 ml of water/kg body Weight  

    maintained by continuous infusion into jugular vein of 2.5% glucose solution and 0.58% NaCl solution at 0.5 ml/min per kg body weight.

 control urine samples are collected by urethral catheter.

Ø Blood samples are obtained in the middle of each clearance period.

Ø After the control period, compounds to be tested are administered and further clearance tests are performed

2) Hydropenia:

Withdrawing the drinking water 48 h before experiment.  0.5 U/kg of vasopressin in oil is injected i.m. before 24 h.

On the day of the experiment 20 mU/kg vasopressin is injected i.v., followed by infusion of 50 mU/kg per hour vasopressin. 

To obtain constant urine flow 5% NaCl solution is infused up to i.v. administration of a compound to be tested, followed by i.v. infusion of 0.9% NaCl solution at a rate equal to the urine flow.

Urine and blood samples are collected.

Ø Glomerular filtration rate (GFR) and renal plasma flow (RPF) are measured by the clearance of inulin and para-aminohippurate, respectively.

Ø Therefore, appropriate infusion of inulin (bolus of 0.08 g/kg followed by infusion of 1.5 mg/kg per min) and para-aminohippurate (bolus 0.04 g/kg followed by infusion of 0.3 mg/kg per min) are initiated

EVALUATION:

Ø The following parameters may be determined:

•      water and electrolyte excretion,

•      GFR= Inulin is used,

•      RPF = para amino hyppurate is used,

•      C_H2O & T_CH20 and plasma renin activity.

Results of test compound are compared statistically with control and standard drug treated animals.

v Free water clearance:  C_H2O

       Amount of urine excreted in excess that needed to clear Salt.

            C_H2O = V – Cosm

v Free water reabsorption T_CH20

      In the presence of ADH urine is concentrated at that time V<Cosm

            T_CH20 = Cosm – V

v Osmolar clearance: Cosm

 Volume of urine containing the solute at the osmolal conc. Equal to that of plasma (Posm)

Cosm = V (Uosm/Posm)

Where,

   V    : urine flow

Uosm :urine osmolarity

Posm :plasma osmolarity. 

5)   MICROPUNCTURE TECHNIQUES

PRINCIPLE:

Ø Effect of diuretics on single nephron function.

Ø Measures changes in tubular fluid re-absorptive rates and electrolyte concentrations can be used to asses the mechanism of action.

PROCEDURE: 

ü Studies are performed in rats with a body weight of about 250 g

ü Anaesthetized by the intraperitoneal inj. of thiopentone/pentobarbital.

ü Rats are fasted for 16 h and After anesthesia the animals are placed on a thermostatically heated table.

ü Tracheotomy is performed

ü carotid artery is cannulated for blood pressure recording

ü jugular vein are cannulated for infusion of compounds

ü Femoral artery is catheterized for obtaining blood.

ü The left kidney is carefully exposed by a flank incision, embedded in a small plastic vessel with cotton wool, and bathed with paraffin oil at 37 °C.

ü The ureter is cannulated to collect urine and rectal temperature monitored continuously.

ü A bolus injection of 75 μCi inulin 3H in 0.7 ml NaCl solution is given, followed by 0.85% NaCl solution at a rate of 2.5 ml/min per 100 g body weight.

Ø After 45 mins. control puncture of tubules is performed.

Ø tubular fluid samples from proximal and distal tubules is collected with glass capillaries (micropipette).

Ø The control period is followed by the test period. After an equilibration period of 30 min with the compound to be tested, micropuncture is performed again and tubular fluid is collected.

The urethral urine is collected and blood sampling is performed.

EVALUATION

The following parameters may be determined:

•      Inulin clearance (GFR),

•      single nephron GFR,

•      fractional delivery of water, Na+ and K+ in proximal and distal tubules and in urine.

All data are expressed as mean values ±SEM(standard error of mean). Comparison of the effects of compounds to be tested with controls is performed by one way analysis of variance and by Student’s t-test for paired and unpaired data.

In vitro Methods

     1)   ISOLATED TUBULE PREPARATION

PURPOSE:

Ø The various tubule segments: proximal tubule (PT); descending thin limb of the loop of Henle(DTL); ascending thin limb of the loop of Henle (ATL); thick ascending limb of the loop of Henle (TAL);  Distal convoluted tubule (DCT);  medullary collecting duct (MCD);  papillary collecting duct (PCD) have different functional properties.

Ø If one has to identify the site and the mechanism of action of a pharmacological agent which acts on kidney function in clearance and micropuncture studies.

PROCEDURE:

Ø Kidney tubule segments of several species like rat, mouse, rabbit used.

Ø The tubule segments are dissected from thin kidney slices (<1 mm thick ,at 4 °C in a Ringer type solution).

Ø The dissected segment is transferred into the perfusion chamber.

Tubule segment perfused with Micropipette 

Ø To perfuse a suitable tubule, one end of the tubule is holded by micropipette

Ø A perfusion pipette is inserted into tubule lumen

Ø The other end of the tubule is sucked into collecting pipette

Ø The oil inside the collecting pipette prevents the evaporation.

Ø All the accumulated fluid is collected at the periodic intervals by inserting a narrow caliberated pipette in the collecting pipette

Ø To approximate the in vivo situation, an isotonic rabbit serum is perfused while the tubule is immersed in a bath of rabbit serum.

EVALUATION:

Ø The absolute value of reabsorption from the change in the concentration of an impermeable marker like inulin and isothalamate in the collecting fluid.

Ø Leaks around the perfusion pipette is detected from the appearance of the marker in the external bath.

2) CARBONIC ANHYDRASE INHIBITION

PRINCIPLE:

Ø Carbonic anhydrase is a zinc-containing enzyme that catalyzes the reversible hydration (or hydroxylation) of CO2 to form H2CO3 which dissociates non-enzymatically into HCO3 – and H+

Ø The enzyme source are red cells, a rich source of the same isoenzymes found in the eye (used in glaucoma also).

PROCEDURE:

Assay --

Ø Here the reaction vessel is used.

Ø CO2 flow rate is adjusted to 30-45ml/min.

In a reaction vessels ,mix Phenol red indicator, 12.5 mg phenol red/liter 2.6 mM NaHCO3, pH 8.3, + 218 mM Na2CO3, CA enzyme From dog blood, 1 M sodium carbonate/bicarbonate buffer, pH 9.8, Water/ Water+ drug

The following parameters are determined in duplicate samples:

Tu = (uncatalyzed time ) = time for the color change to occur in the absence of    enzyme.

Te = (catalyzed time) = time for the color change to occur in the presence of the enzyme.

Tu – Te = enzyme rate

Ti = enzyme rate in the presence of various concentrations of inhibitor

EVALUATION:

Percent inhibition of carbonic anhydrase is calculated according to the following formula:

3) PATCH CLAMP TECHNIQUE

PRINCIPLE:

Ø This technique allow the study of single ion channels as well as whole cell ion channel currents.

Ø It requires patch electrode with relatively large tip (>1mm)that has smooth surface .

PROCEDURE:

Ø Technique can be applied to cultured kidney cells, to freshly isolated kidney cells or to cells of isolated perfused kidney tubules.

Cell membrane with patch-clamp electrode 

Ø Patch clamp electrode is passed against a cell membrane and suction is applied to pull the cell membrane inside the electrode tip.

Ø The suction causes cell to form tight, high resistance seal with rim of electrode, usually greater than 10 gigaOhms which is called a gigaseal.

Ø Cell attached, whole cell mode, inside out mode, outside out mode of this technique allow investigation of ion channels.

Different mode of techniques for determination of ion channels

EVALUATION :

v Concentration response curve of the drug which inhibits ion channel can be obtained.

v Single ion channels studied by cell attached technique

v Co transport system is studied by whole cell patch clamp technique as transport rate of single event is too small to detect.